PROJECT SUMMARY The highest known genetic risk of schizophrenia (SZ) is conferred by hemizygous microdeletion of chromosome 22q11. The proline dehydrogenase gene (PRODH) is located in the common deleted region, and encodes the enzyme that catalyzes proline catabolism. Proline is a neuromodulator at glutamatergic synapses, and the peripheral hyperprolinemia arising from PRODH mutations or CNVs encompassing PRODH, has been associated with cognitive impairment and decreased IQ. We recently reported a highly significant association of hyperprolinemia with SZ. We now provide evidence that two further independent SZ risk factors: Low vitamin-D status, and mutation of the dysbindin gene (Dtnbp1), also cause hyperprolinemia. Our findings suggest convergence of the biological pathways regulated by vitamin-D and DTNBP1, via loss of PRODH expression, with elevated proline as a common SZ endophenotype. Studies have documented the dysfunctional consequences of hyperprolinemia, such as aberrant glutamatergic and dopamine signaling leading to, for example, cognitive deficits and attenuated prepulse inhibition (PPI). Targeting hyperprolinemia in the Dtnbp1 model may therefore positively impact neurotransmitter signaling and restore function. This proposed study is designed to test our hypotheses under the following Specific Aims. Aim 1: To test the relative molecular contributions of low Vitamin D, and PRODH, and DTNBP1 gene variants, to SZ- associated hyperprolinemia. In a SZ patient and control sample (n=250), we will measure plasma proline and Vitamin-D levels, confirming the strong relationship between elevated proline and low Vitamin-D. From our preliminary data, we anticipate that 80% of the measured hyperprolinemia will arise from low vitamin-D, while DTNBP1 and PRODH variants will be responsible for the remaining 20%. Thus, we will perform molecular analyses on our complete study cohort (404 subjects), screening for PRODH, and DTNBP1 variants that alter gene expression, and testing for variant associations, plus interactions with low Vitamin-D, on the outcome of proline elevation. Aim 2: To examine the molecular pathway leading to hyperprolinemia in the Dtnbp1 model. Aim 2A. Molecular analyses will include assay of peripheral and CNS tissue expression of Prodh, p53 regulated genes, and Comt (downstream of Prodh), as well as of cortical, hippocampal and peripheral proline levels. Aim 2B. Utilizing primary neurons and astrocytes from sdy-/- and Prodh+/- mice, we will directly upregulate Prodh expression, via treatment with Vitamin D and the thiazolidinedione drug Rosiglitazone (RZG), testing for restoration of Prodh expression and decreased cellular proline. Aim 3: To target hyperprolinemia in the Dtnbp1 model in vivo. We will examine whether treatment with Vitamin D (Aim 3a), RZG (Aim 3b), and, to seek to reduce the impact of off-target effects, a proline-deficient diet (Aim 3c), compared to vehicle- treatment alone, restores the above pathway in vivo, and prevents or alleviates the Sdy-/- deficit in, for example, PPI and spatial working memory.